A 7-year-old Chinese boy with a history of bronchial asthma had upper respiratory tract infection for the preceding two weeks treated by a pediatrician. He then presented with confusion and incoherent speech on the day of admission. He was afebrile, with the Glasgow coma scale (GCS) of 14/15 (E4V4M6) and normal physical findings. Investigations showed normal complete blood counts, blood glucose, renal and liver function tests except for mildly elevated ALT at 34 IU/L (5-25 IU/L). His blood ammonia, lactate and pyruvate levels were normal. The CT brain showed no space occupying lesion or cerebral edema. Lumbar puncture showed an opening pressure of 12cm water, CSF glucose 3.1mmol/L, protein 1.25 gm/L (0.15-0.45 gm/L), RBC 2/mm3 and WBC 2/mm3. The gram stain showed no organisms. The nasopharyngeal aspirate for rapid antigen test for both influenza A and B were negative. About 12 hr after admission, he developed generalized tonic convulsions progressing to decorticate posturing, GCS 3/15 (E1V1M1) and pupils 2.5mm, equal and reacting sluggishly to light. Seizures were controlled with anticonvulsants. He was intubated and put on mechanical ventilation. Despite treatment with acyclovir, cefotaxime, anti-tuberculous medications, dexamethasone, hyperventilation and mannitol infusion, the child remained comatose. MRI of brain was done (see below). His condition continued to deteriorate and the pupils became fixed and dilated 34 hours after hospitalization. He finally succumbed on the fourth day of hospitalization.

Subsequent laboratory reports showed that the nasopharyngeal aspirate and throat swab taken on the second day of hospitalization were positive for Influenza A H1N1 by culture (human strain influenza A H1N1/Brisbane/59/2007). Complement fixing antibody titer against influenza A nucleoprotein showed at least a 8-fold rise within 3 days but hemagglutinating antibody titer was high only for H3. The serological findings were compatible with a recent Influenza A H3 infection while the culture confirmed a current H1N1 infection. Further testing of the child's serum was done after the autopsy findings were available. These showed negative anti-neutrophil cytoplasmic antibody (ANCA), positive anti-nuclear antibody at 1/240 and 1/80 with a homogenous pattern, normal anti-double stranded DNA titer, negative anti-ribonucleoprotein antibody and non-diagnostic anti-cardiolipin antibody level. Overall, there was no serological evidence of a rheumatic disease, in particular systemic lupus erythematosus. The serum interleukin 6 was elevated to 7.01 pg/mL on admission (reference < 5.9) and progressively raised to 130pg/mL on the day of death. The serum tumor necrosis factor alpha was also persistently raised in the range of 17.8-15.0 pg/mL (reference < 8.1).

Extra-cranial organs were remarkable only for slightly heavier lungs (left lung 190 gm, reference 166 gm; right lung 250 gm, reference 177 gm) and congested airways. In particular, the liver was normal. Microscopy showed focal lymphocytic bronchitis and bronchiolitis, focal alveolar edema and hyperplastic smooth muscle around the bronchi and bronchioles. These changes were compatible with the history of intercurrent viral infection and bronchial asthma. Assessment of steatosis by frozen section and oil-red-O stain showed moderate steatosis with fine and medium sized fat globules in the liver but no steatosis was noted in the heart, kidney, skeletal muscle and brain.

The brain was heavy (1,700 gm; reference 1,290 gm), markedly congested and edematous. Cerebellar tonsillar and early bilateral uncal herniations were noted. The dural veins and the Circle of Willis were normal. The lateral ventricles were slit-like. There was symmetrical hemorrhagic necrosis or softening of the head of caudate nucleus, the corpus callosum, the periventricular white matter, the lateral and posterior part of the thalamus and the internal capsule. Figures 2, 3 and 4 show coronal sections of cerebrum at the genu of the corpus callosum, mamillary bodies and splenium of the corpus callosum respectively. There was artifactual disruption of the right temporal lobes as shown in Figure 3 and 4. The cortical grey matter, the major part of the centrum semiovale, putamen and globus pallidus showed no gross lesions. The brainstem, cerebellum and spinal cord were pale, edematous and focally congested.

On microscopy, necrosis and vasculopathy were noted. The common vasculopathy was an exudative vasculopathy (EV) characterized by an expanded perivascular space with fibrinous exudate, often with a vacuolated artifact. (Figure 5: arteriole about 250 m in diameter in right thalamus.) EV involved small vessels and arterioles distributed throughout the brain including the necrotic and non-necrotic areas. The other forms of vasculopathy were necrotizing vasculopathy (NV) and fibrinous vasculitis (FVL), both restricted to the necrotic areas only. NV resembled EV except for the presence of fibrinous necrosis of the vessel wall. (Figure 6: 110 m vessel in the left head of caudate.) FVL showed additional lymphocytic and neutrophilic exudate on the vascular wall and perivascular space and karyorrhexis with or without luminal thrombosis. (Figure 7: 90 m vessel in the left head of caudate, Figure 8: 50 m vessel in the right head of caudate, Figure 9: 80 m vessel in right internal capsule.) Fibrin deposits in the vessels were confirmed by Martius-scarlet-blue stain (Figure 10: same vessel as Figure 9) and immunohistochemistry for fibrin (Figure 11: same vessel as Figure 9). No perivascular demyelination was evident (Figure 12: Luxol fast blue stain, same vessel as Figure 9). The necrotic areas showed ghost shadow of necrotic cells, pyknotic or karyorrhectic nuclear fragments and extravasated red cells in a background of paucicellular and edematous neuropil. There was neither astrocytic nor microglial proliferation. Many of the neurons in the cerebral cortex, hippocampus, cerebellum and brainstem showed ischemic shrinkage and pericellular edema. Some small vessels in the brainstem also showed thrombosis. The generalized ischemic changes of the neurons, thrombosis in the brainstem and cerebellar coning were considered pathological changes secondary to the cerebral edema. The generalized cerebral edema, vasculopathies and necrosis were considered the primary pathology.
RT-PCR was done on fresh samples of the cerebrum, pons, heart, lung, skeletal muscle, spleen, intestine and kidney for influenza A and B, respiratory syncytial virus, parainfluenza 1, 2, 3 and 4, adenovirus, rhinovirus, human metapneumovirus, enterovirus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus and varizella-zoster virus and the results were all negative. Viral cell cultures of these tissues were also negative.